Engineered mutant a-ENaC subunit mRNA delivered by lipid nanoparticles reduces amiloride currents in cystic fibrosis - based cell and mice models
Research performed at REU - Oregon Health & Sciences University CDCB Summer Internship
Cystic fibrosis (CF) is a devastating disorder that affects 70,000 patients worldwide. Morbidity in CF is mainly caused by thick airway mucus formation, lung infection, and terminal pulmonary deterioration. Mutations in the cystic fibrosis transmembrane regulator (CFTR) gene decrease either expression or activity of CFTR protein causing CF. Current therapies are effective for certain classes of CFTR mutations only, leaving approximately 30% of patients without treatment options. In the airway, defective CFTR protein fails to secrete Cl- leading to isotonic Na+ hyperabsorption by the Epithelial Sodium Channel (ENaC). This causes reduction in the airway surface liquid (ASL) height and impediment of mucociliary clearance. Thus, ENaC inhibition can potentially reverse ASL dehydration and be an effective therapy for all CF patients, independent of the underlying class of CFTR mutation. Previous attempts at pharmacological or RNAi-based ENaC inhibition were unsuccessful in clinical trials due to off-target effects. ENaC is composed of three homologous α, β, and γ subunits with the α-ENaC subunit being essential in functional channel formation. Our approach: Competitive inhibition of endogenous ENaC by transfecting inactive α ENaC subunit mRNA. We hypothesized: “Delivery of nonfunctional α ENaC mRNA would decrease ENaC hyperactivity in vitro and in vivo.” To test the effect of αmutENaC transfection in vitro, polarized cystic fibrosis bronchial epithelial cells were used and amiloride-sensitive short circuit ENaC current was measured. We found that transfected cells had a significant decrease in macroscopic as well as ENaC currents compared to untransfected controls. Similarly, nasal instillation of αmutENaC in CFTR null mice resulted in a decrease in amiloride sensitive nasal potential difference in vivo. In conclusion, our data indicates that mRNA-based intervention can effectively inhibit endogenous ENaC activity. We speculate that ENaC inhibition will increase ASL height and improve mucociliary clearance in vitro and in vivo.
Biology Department; Chemistry Department; Biochemistry Department